protein RNA The
Chapter 17.
From Gene to Protein AP Biology
2005-2006
Metabolism teaches us about genes
Metabolic defects
studying metabolic diseases suggested that genes specified proteins
A AP Biology
alkaptonuria (black urine from alkapton) PKU (phenylketonuria) Genes create phenotype
each disease is caused by non-functional enzyme
B
C
D
E 2005-2006
AP Biology
2005-2006
1 gene – 1 enzyme hypothesis
Beadle & Tatum
Compared mutants of bread mold, Neurospora fungus
created mutations by X-ray treatments X-rays break DNA inactivate a gene
wild type grows on “minimal” media sugars + required precursor nutrient to synthesize essential amino acids
mutants require added amino acids each type of mutant lacks a certain enzyme
AP Biology
needed to produce a certain amino acid non-functional enzyme = broken gene
2005-2006
1941 | 1958
Beadle & Tatum
George Beadle
Edward Tatum AP Biology
2005-2006
Beadle & Tatum’s Neurospora experiment
AP Biology
2005-2006
So… What is a gene?
One gene – one enzyme
One gene – one protein
but many proteins are composed of several polypeptides but each polypeptide has its own gene
One gene – one polypeptide
but not all proteins are enzymes but all proteins are coded by genes
but many genes only code for RNA
One gene – one product
AP Biology
but many genes code for more than one product …
Where does that leave us?! 2005-2006
Defining a gene… “Defining a gene is problematic because… one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.” RNA gene – Elizabeth Pennisi, Science 2003 polypeptide 1
gene
polypeptide 2
AP Biology
polypeptide 3
It’s hard to hunt for wabbits, if you don’t know what a wabbit looks like.
2005-2006
The “Central Dogma”
How do we move information from DNA to proteins? transcription
DNA
replication
AP Biology
translation
RNA
protein For simplicity sake, let’s go back to genes that code for proteins…
2005-2006
From nucleus to cytoplasm…
Where are the genes?
Where are proteins synthesized?
genes are on chromosomes in nucleus proteins made in cytoplasm by ribosomes
How does the information get from nucleus to cytoplasm?
AP Biology
messenger RNA
nucleus
2005-2006
RNA
ribose sugar N-bases uracil instead of thymine U : A C : G
single stranded mRNA, rRNA, tRNA, siRNA…. transcription
DNA AP Biology
RNA
2005-2006
Transcription
Transcribed DNA strand = template strand
Synthesis of complementary RNA strand
untranscribed DNA strand = coding strand transcription bubble
Enzyme
AP Biology
RNA polymerase
2005-2006
Transcription in Prokaryotes
Initiation
RNA polymerase binds to promoter sequence on DNA
Role of promoter 1. Where to start reading = starting point 2. Which strand to read = template strand 3. Direction on DNA = always AP Biology reads DNA 3'5'
2005-2006
Transcription in Prokaryotes
Promoter sequences
AP Biology
RNA polymerase molecules bound to bacterial DNA 2005-2006
Transcription in Prokaryotes
Elongation RNA polymerase unwinds DNA ~20 base pairs at a time reads DNA 3’5’ builds RNA 5’3’ (the energy governs the synthesis!)
No proofreading 1 error/105 bases many copies short life not worth it!
AP Biology
2005-2006
Transcription
RNA
AP Biology
2005-2006
Transcription in Prokaryotes
Termination RNA polymerase stops at termination sequence mRNA leaves nucleus through pores
RNA GC hairpin turn
AP Biology
2005-2006
Transcription in Eukaryotes
AP Biology
2005-2006
Prokaryote vs. Eukaryote genes
Prokaryotes
Eukaryotes
DNA in cytoplasm circular chromosome naked DNA
no introns
DNA in nucleus linear chromosomes DNA wound on histone proteins introns vs. exons
intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence AP Biology
2005-2006
Transcription in Eukaryotes
3 RNA polymerase enzymes
RNA polymerase I
RNA polymerase I I
AP Biology
transcribes genes into mRNA
RNA polymerase I I I
only transcribes rRNA genes
only transcribes rRNA genes
each has a specific promoter sequence it recognizes 2005-2006
Transcription in Eukaryotes
Initiation complex
transcription factors bind to promoter region upstream of gene
AP Biology
proteins which bind to DNA & turn on or off transcription TATA box binding site
only then does RNA polymerase bind to DNA 2005-2006
Post-transcriptional processing
Primary transcript
eukaryotic mRNA needs work after transcription
Protect mRNA
from RNase enzymes in cytoplasm add 5' cap mRNA 5' cap add polyA tail 5' G PPP CH
3' A
3
Edit out introns intron = noncoding (inbetween) sequence
eukaryotic DNA exon = coding (expressed) sequence
primary mRNA transcript AP Biology
mature mRNA transcript
pre-mRNA
2005-2006 spliced mRNA
Transcription to translation
Differences between prokaryotes & eukaryotes time & physical separation between processes RNA processing
AP Biology
2005-2006
Translation in Prokaryotes
Transcription & translation are simultaneous in bacteria DNA is in cytoplasm no mRNA editing needed
AP Biology
2005-2006
From gene to protein
transcription
DNA
mRNA
mRNA leaves nucleus through nuclear pores
AP Biology
nucleus
translation
a a
a a
protein
a ribosomea
cytoplasm
a a
a a
a a
a a
a a
proteins synthesized by ribosomes using 2005-2006 instructions on mRNA
How does mRNA code for proteins? DNA
TACGCACATTTACGTACGCGG
mRNA AUGCGUGUAAAUGCAUGCGCC
? protein
AP Biology
MetArg ValAsn AlaCys Ala How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)? 2005-2006
Cracking the code
1960 | 1968
Nirenberg & Matthaei
determined 1st codon–amino acid match
UUU coded for phenylalanine
created artificial poly(U) mRNA added mRNA to test tube of ribosomes, tRNA & amino acids
mRNA synthesized single amino acid polypeptide chain
phe–phe–phe–phe–phe–phe
AP Biology
2005-2006
AP Biology
Heinrich Matthaei
Marshall2005-2006 Nirenberg
Translation
Codons
AP Biology
blocks of 3 nucleotides decoded into the sequence of amino acids
2005-2006
mRNA codes for proteins in triplets DNA
TACGCACATTTACGTACGCGG
mRNA AUGCGUGUAAAUGCAUGCGCC
? protein
AP Biology
MetArg ValAsn AlaCys Ala
2005-2006
The code
For ALL life!
strongest support for a common origin for all life
Code is redundant
several codons for each amino acid
Why is this a good thing?
Start codon
AUG methionine
Stop codons
UGA, AP Biology
UAA, UAG
2005-2006
How are the codons matched to amino acids? DNA
3'
5'
5'
3'
TACGCACATTTACGTACGCGG
mRNA AUGCGUGUAAAUGCAUGCGCC codon 3'
5'
tRNA
UAC
amino acid
Met
AP Biology
GCA Arg
CAU anti-codon Val
2005-2006
aa aa aa aa
cytoplasm aa
transcription
translation
aa aa aa
protein aa aa
aa
nucleus AP Biology
2005-2006
tRNA structure
“Clover leaf” structure anticodon on “clover leaf” end amino acid attached on 3' end
AP Biology
2005-2006
Loading tRNA
Aminoacyl tRNA synthetase enzyme which bonds amino acid to tRNA endergonic reaction
energy stored in tRNA-amino acid bond
AP Biology
ATP AMP
unstable so it can release amino acid at ribosome 2005-2006
Ribosomes
Facilitate coupling of tRNA anticodon to mRNA codon
organelle or enzyme?
Structure ribosomal RNA (rRNA) & proteins 2 subunits
AP Biology
large small 2005-2006
Ribosomes
P site (peptidyl-tRNA site)
A site (aminoacyl-tRNA site)
holds tRNA carrying growing polypeptide chain holds tRNA carrying next amino acid to be added to chain
E site (exit site)
AP Biology
empty tRNA leaves ribosome from exit site 2005-2006
Building a polypeptide
Initiation
brings together mRNA, ribosome subunits, proteins & initiator tRNA
Elongation Termination
AP Biology
2005-2006
Elongation: growing a polypeptide
AP Biology
2005-2006
Termination: release polypeptide
Release factor “release protein” bonds to A site bonds water molecule to polypeptide chain
Now what happens to the polypeptide?
AP Biology
2005-2006
Protein targeting
Signal peptide
address label
Destinations:
secretion nucleus mitochondria chloroplasts cell membrane cytoplasm
start of a secretory pathway
AP Biology
2005-2006
RNA polymerase
DNA
Can you tell the story?
amino acids exon
intron
tRNA
pre-mRNA
5' cap
mature mRNA aminoacyl tRNA synthetase
polyA tail large subunit
polypeptide
ribosome
5' small subunit
AP Biology
tRNA E P A
2005-2006
3'
Put it all together…
AP Biology
2005-2006
Any Questions??
AP Biology
2005-2006
From Gene to Protein AP Biology
2005-2006
Metabolism teaches us about genes
Metabolic defects
studying metabolic diseases suggested that genes specified proteins
A AP Biology
alkaptonuria (black urine from alkapton) PKU (phenylketonuria) Genes create phenotype
each disease is caused by non-functional enzyme
B
C
D
E 2005-2006
AP Biology
2005-2006
1 gene – 1 enzyme hypothesis
Beadle & Tatum
Compared mutants of bread mold, Neurospora fungus
created mutations by X-ray treatments X-rays break DNA inactivate a gene
wild type grows on “minimal” media sugars + required precursor nutrient to synthesize essential amino acids
mutants require added amino acids each type of mutant lacks a certain enzyme
AP Biology
needed to produce a certain amino acid non-functional enzyme = broken gene
2005-2006
1941 | 1958
Beadle & Tatum
George Beadle
Edward Tatum AP Biology
2005-2006
Beadle & Tatum’s Neurospora experiment
AP Biology
2005-2006
So… What is a gene?
One gene – one enzyme
One gene – one protein
but many proteins are composed of several polypeptides but each polypeptide has its own gene
One gene – one polypeptide
but not all proteins are enzymes but all proteins are coded by genes
but many genes only code for RNA
One gene – one product
AP Biology
but many genes code for more than one product …
Where does that leave us?! 2005-2006
Defining a gene… “Defining a gene is problematic because… one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.” RNA gene – Elizabeth Pennisi, Science 2003 polypeptide 1
gene
polypeptide 2
AP Biology
polypeptide 3
It’s hard to hunt for wabbits, if you don’t know what a wabbit looks like.
2005-2006
The “Central Dogma”
How do we move information from DNA to proteins? transcription
DNA
replication
AP Biology
translation
RNA
protein For simplicity sake, let’s go back to genes that code for proteins…
2005-2006
From nucleus to cytoplasm…
Where are the genes?
Where are proteins synthesized?
genes are on chromosomes in nucleus proteins made in cytoplasm by ribosomes
How does the information get from nucleus to cytoplasm?
AP Biology
messenger RNA
nucleus
2005-2006
RNA
ribose sugar N-bases uracil instead of thymine U : A C : G
single stranded mRNA, rRNA, tRNA, siRNA…. transcription
DNA AP Biology
RNA
2005-2006
Transcription
Transcribed DNA strand = template strand
Synthesis of complementary RNA strand
untranscribed DNA strand = coding strand transcription bubble
Enzyme
AP Biology
RNA polymerase
2005-2006
Transcription in Prokaryotes
Initiation
RNA polymerase binds to promoter sequence on DNA
Role of promoter 1. Where to start reading = starting point 2. Which strand to read = template strand 3. Direction on DNA = always AP Biology reads DNA 3'5'
2005-2006
Transcription in Prokaryotes
Promoter sequences
AP Biology
RNA polymerase molecules bound to bacterial DNA 2005-2006
Transcription in Prokaryotes
Elongation RNA polymerase unwinds DNA ~20 base pairs at a time reads DNA 3’5’ builds RNA 5’3’ (the energy governs the synthesis!)
No proofreading 1 error/105 bases many copies short life not worth it!
AP Biology
2005-2006
Transcription
RNA
AP Biology
2005-2006
Transcription in Prokaryotes
Termination RNA polymerase stops at termination sequence mRNA leaves nucleus through pores
RNA GC hairpin turn
AP Biology
2005-2006
Transcription in Eukaryotes
AP Biology
2005-2006
Prokaryote vs. Eukaryote genes
Prokaryotes
Eukaryotes
DNA in cytoplasm circular chromosome naked DNA
no introns
DNA in nucleus linear chromosomes DNA wound on histone proteins introns vs. exons
intron = noncoding (inbetween) sequence eukaryotic DNA exon = coding (expressed) sequence AP Biology
2005-2006
Transcription in Eukaryotes
3 RNA polymerase enzymes
RNA polymerase I
RNA polymerase I I
AP Biology
transcribes genes into mRNA
RNA polymerase I I I
only transcribes rRNA genes
only transcribes rRNA genes
each has a specific promoter sequence it recognizes 2005-2006
Transcription in Eukaryotes
Initiation complex
transcription factors bind to promoter region upstream of gene
AP Biology
proteins which bind to DNA & turn on or off transcription TATA box binding site
only then does RNA polymerase bind to DNA 2005-2006
Post-transcriptional processing
Primary transcript
eukaryotic mRNA needs work after transcription
Protect mRNA
from RNase enzymes in cytoplasm add 5' cap mRNA 5' cap add polyA tail 5' G PPP CH
3' A
3
Edit out introns intron = noncoding (inbetween) sequence
eukaryotic DNA exon = coding (expressed) sequence
primary mRNA transcript AP Biology
mature mRNA transcript
pre-mRNA
2005-2006 spliced mRNA
Transcription to translation
Differences between prokaryotes & eukaryotes time & physical separation between processes RNA processing
AP Biology
2005-2006
Translation in Prokaryotes
Transcription & translation are simultaneous in bacteria DNA is in cytoplasm no mRNA editing needed
AP Biology
2005-2006
From gene to protein
transcription
DNA
mRNA
mRNA leaves nucleus through nuclear pores
AP Biology
nucleus
translation
a a
a a
protein
a ribosomea
cytoplasm
a a
a a
a a
a a
a a
proteins synthesized by ribosomes using 2005-2006 instructions on mRNA
How does mRNA code for proteins? DNA
TACGCACATTTACGTACGCGG
mRNA AUGCGUGUAAAUGCAUGCGCC
? protein
AP Biology
MetArg ValAsn AlaCys Ala How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)? 2005-2006
Cracking the code
1960 | 1968
Nirenberg & Matthaei
determined 1st codon–amino acid match
UUU coded for phenylalanine
created artificial poly(U) mRNA added mRNA to test tube of ribosomes, tRNA & amino acids
mRNA synthesized single amino acid polypeptide chain
phe–phe–phe–phe–phe–phe
AP Biology
2005-2006
AP Biology
Heinrich Matthaei
Marshall2005-2006 Nirenberg
Translation
Codons
AP Biology
blocks of 3 nucleotides decoded into the sequence of amino acids
2005-2006
mRNA codes for proteins in triplets DNA
TACGCACATTTACGTACGCGG
mRNA AUGCGUGUAAAUGCAUGCGCC
? protein
AP Biology
MetArg ValAsn AlaCys Ala
2005-2006
The code
For ALL life!
strongest support for a common origin for all life
Code is redundant
several codons for each amino acid
Why is this a good thing?
Start codon
AUG methionine
Stop codons
UGA, AP Biology
UAA, UAG
2005-2006
How are the codons matched to amino acids? DNA
3'
5'
5'
3'
TACGCACATTTACGTACGCGG
mRNA AUGCGUGUAAAUGCAUGCGCC codon 3'
5'
tRNA
UAC
amino acid
Met
AP Biology
GCA Arg
CAU anti-codon Val
2005-2006
aa aa aa aa
cytoplasm aa
transcription
translation
aa aa aa
protein aa aa
aa
nucleus AP Biology
2005-2006
tRNA structure
“Clover leaf” structure anticodon on “clover leaf” end amino acid attached on 3' end
AP Biology
2005-2006
Loading tRNA
Aminoacyl tRNA synthetase enzyme which bonds amino acid to tRNA endergonic reaction
energy stored in tRNA-amino acid bond
AP Biology
ATP AMP
unstable so it can release amino acid at ribosome 2005-2006
Ribosomes
Facilitate coupling of tRNA anticodon to mRNA codon
organelle or enzyme?
Structure ribosomal RNA (rRNA) & proteins 2 subunits
AP Biology
large small 2005-2006
Ribosomes
P site (peptidyl-tRNA site)
A site (aminoacyl-tRNA site)
holds tRNA carrying growing polypeptide chain holds tRNA carrying next amino acid to be added to chain
E site (exit site)
AP Biology
empty tRNA leaves ribosome from exit site 2005-2006
Building a polypeptide
Initiation
brings together mRNA, ribosome subunits, proteins & initiator tRNA
Elongation Termination
AP Biology
2005-2006
Elongation: growing a polypeptide
AP Biology
2005-2006
Termination: release polypeptide
Release factor “release protein” bonds to A site bonds water molecule to polypeptide chain
Now what happens to the polypeptide?
AP Biology
2005-2006
Protein targeting
Signal peptide
address label
Destinations:
secretion nucleus mitochondria chloroplasts cell membrane cytoplasm
start of a secretory pathway
AP Biology
2005-2006
RNA polymerase
DNA
Can you tell the story?
amino acids exon
intron
tRNA
pre-mRNA
5' cap
mature mRNA aminoacyl tRNA synthetase
polyA tail large subunit
polypeptide
ribosome
5' small subunit
AP Biology
tRNA E P A
2005-2006
3'
Put it all together…
AP Biology
2005-2006
Any Questions??
AP Biology
2005-2006
